Chronic exposure to tobacco smoke accounts for ~5 million deaths per year making health complications from smoking the primary cause of preventable mortality in the world. Nicotine, the addictive component of tobacco, binds to and activates neuronal nicotinic acetylcholine receptors (nAChRs), ligand-gated cation channels that are normally activated by the endogenous neurotransmitter, acetylcholine. Nicotine initiates dependence by activating neurons within the ventral tegmental area (VTA) of the mesocorticolimbic reward circuitry, ultimately driving the release of dopamine (DA) within the nucleus accumbens, a phenomenon widely associated with the rewarding or reinforcing value of nicotine. A large variety of nAChR subunit genes are expressed in both VTA DAergic projection neurons and GABAergic interneurons but activation of nAChRs containing alpha4 and beta2 subunits in the VTA are most critical for the rewarding properties of nicotine. Exposure to chronic nicotine functionally upregulates nAChRs in various brain regions although the behavioral consequence of this upregulation is unknown. In the midbrain, nAChRs containing the alpha4 subunit are selectively upregulated in GABAergic neurons but not DAergic neurons. The goal of this R21 proposal is to gain insight into how this neuronal subtype selective functional upregulation of the alpha4 subunit affects nicotine reward behavior. Specific aim one will use viral-mediated gene delivery to selectively express gain-of-function alpha4 subunits in VTA GABAergic neurons of mice and measure nicotine-mediated reward behavior compared to control animals. Specific aim two will use immunohistochemical and biophysical approaches to determine how increased activation of GABAergic neurons by nicotine affects VTA DAergic neuron activity. The results from these studies will not only help define the role of GABAergic neurons in nicotine reward, but should also unmask behavioral consequences of nAChR upregulation.